Distinct and identifiable populations of regulatory T cells control the physiological outgrowth and function of autoreactive cells in peripheral tissues. In animal models, loss or dysfunction of regulatory T cells can lead to autoimmune disease. We have identified a unique population of thymus-derived T cell receptor (TCR)+, CD4+ regulatory T cells in murine models of allogeneic bone marrow transplantation (BMT) and donor leukocyte infusion (DLI) therapy that specifically suppress the GVH reactivity of donor T cells. These cells may help establish and maintain peripheral tolerance to host and donor antigens. Regulatory TCR+ CD4+ T cells from DLI-treated mice suppress alloreactivity in an alloantigen-specific manner in vitro and GVH disease after adoptive transfer in vivo. We hypothesize that similar regulatory T cells emerge from the human thymus post-transplant, but that diminished capacity to generate regulatory T cells (due to age, prior chemotherapy, radiation, etc.) contributes to a higher risk of GVH disease after DLI therapy and, possibly, to the onset of chronic GVH disease. Quantitative measurements of TRECs in combination with unique phenotypic markers now allow for a definitive assessment of the kinetics and extent of the thymic contribution to post-transplant immune reconstitution in humans. Using these techniques, we propose to identify and isolate human regulatory T cells in the high TREC-expressing recent thymic emigrant (RTE) population and correlate their presence (or absence) with clinical outcome after allogeneic BMT (with or without DLI therapy). The purpose of this R21 exploratory grant is to generate data in humans that supports our hypothesis and validates the extension of observations made in preclinical models to human BMT. Thymic regulatory T cells may be critical to the success of DLI therapy without GVH disease as well as to the successful induction of tolerance by costimulatory blockade. We propose to determine: (a) whether regulatory T cells can be detected within the population of RTE cells in the peripheral blood of human recipients of HLA-disparate BMT; (b) whether regulatory T cells can be induced and expanded ex vivo from the peripheral blood of BMT patients for possible use in adoptive transfer protocols to prevent GVH disease, and (c) whether induction of anergic T cells by costimulatory blockade ex vivo induces allospecific regulatory cells in either the BMT patient or the donor.